Abstract
DNA replication is a process in which a cell duplicates its genome before cell division,
and must proceed accurately and in organized manner to guarantee maintenance of the
integrity of the genetic information. DNA polymerases are enzymes that catalyse the
synthesis of the new DNA strand by utilizing the parental strand as a template. In
addition to chromosomal replication, DNA synthesis and therefore DNA polymerases are also
needed in other processes like DNA repair and DNA recombination. The DNA polymerase is
an essential DNA polymerase in eukaryotes and is required for chromosomal DNA
replication. It has also been implicated in DNA repair, recombination, and in
transcriptional and cell cycle control. The regulation of the human enzyme was explored
by analysing its expression, phosphorylation and protein-protein interactions.
Expression of both the A and B subunits of the human DNA polymerase ε was strongly
growth-regulated. After serum-stimulation of quiescent fibroblasts, the steady-state mRNA
levels were up-regulated at least 5-fold. In actively cycling cells, however, the
steady-state mRNA and protein levels fluctuated less than 2-fold, being highest in
G1/S phase.
The promoter of the B subunit gene was analysed in detail. The 75 bp core promoter was
essentially dependent on the Sp1 transcription factor. Furthermore, mitogenic control of
the promoter required an intact E2F binding element, and binding of E2F2, E2F4 and p107
was demonstrated in vitro. A down-regulation element, located
immediately downstream from the core promoter, bound E2F1, NF-1 and pRb transcription
factors. A model of the promoter function is presented.
Topoisomerase IIβ binding protein 1 (TopBP1) was found to be associated with human
DNA polymerase ε. TopBP1 contains eight BRCT domains and is homologous to
Saccharomyces cerevisiae Dpb11, Schizosaccharomyces
pombe Cut5, Drosophila melanogaster Mus101 and the human
Breast Cancer susceptibility protein 1 (BRCA1). TopBP1 is a phosphoprotein, whose
expression is induced at the G1/S border and is required for
chromosomal DNA replication. It co-localizes in S phase with BRCA1 into discrete foci,
which do not represent sites of ongoing DNA replication. However, if DNA is damaged or
replication is blocked in S phase cells, TopBP1 and BRCA1 re-localize into proliferating
cell nuclear antigen (PCNA) containing foci that represent stalled replication forks.
Finally, phosphorylation of DNA polymerase ε was described and at least three
immunologically distinct and differentially phosphorylated forms were shown to exist.
Phosphorylation is on serine and threonine residues and shows a cell cycle dependent
fluctuation, but is not affected by DNA damage or by inhibition of DNA replication. BRCA1
co-immunoprecipitates with a hypophosphorylated form of DNA polymerase ε. In
contrast, TopBP1 was shown to be associated with a hyperphosphorylated form.
| Identifer | oai:union.ndltd.org:oulo.fi/oai:oulu.fi:isbn951-42-6581-5 |
| Date | 27 November 2001 |
| Creators | Tuusa, J. (Jussi) |
| Publisher | University of Oulu |
| Source Sets | University of Oulu |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis, info:eu-repo/semantics/publishedVersion |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess, © University of Oulu, 2001 |
| Relation | info:eu-repo/semantics/altIdentifier/pissn/0355-3221, info:eu-repo/semantics/altIdentifier/eissn/1796-2234 |
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